REAGENT or RESOURCE | SOURCE | IDENTIFIER |
---|---|---|
Antibodies | ||
Cleaved Caspase-3 (Asp175) Antibody | Cell Signaling | Cat#9669s; RRID:AB_2341188 |
Bacterial and Virus Strains | ||
E. coli BL21(DE3) | Lucigen | Cat#60401 |
E. coli XL10-Gold Ultracompetent Cells | Agilent | Cat#200315 |
One shot Stbl3 Chemical Competent cells E.coli | ThermoFisher | Cat#C737303 |
Chemicals, Peptides, and Recombinant Proteins | ||
Terrific Broth | Research Products International | Cat#T5100-5000.0 |
Luria Broth | Research Products International | Cat#L24400 |
Isopropyl β-D-1-thiogalactopyranoside (IPTG) | American Bioanalytical | Cat#AB00841-00010 |
NuPAGE LDS Sample Buffer (4X) | Thermo Fisher | Cat#NP0008 |
Dithiothreitol (DTT) | American Bioanalytical | Cat#AB00490-00100 |
SimplyBlue SafeStain | Thermo Fisher | Cat#LC6060 |
KOD Hot Start DNA polymerase | Novagen | Cat#710863 |
Rabbit 40S ribosome | Lomakin and Steitz, 2013 | N/A |
Recombinant Nsp1 | This paper | N/A |
Recombinant eIF3j | This paper | N/A |
DPBS, no calcium, no magnesium | GIBCO | Cat#14190250 |
DMEM, high glucose,pyruvate | GiBCO | Cat#11995065 |
Fetal Bovine Serum | Corning | Cat#35-011-CV |
Penicillin-Streptomycin (10,000 U/mL) | GIBCO | Cat#15140122 |
QIAquick gel extraction KIt | QIAGEN | Cat#28706 |
E-Gel Low Range Quantitative DNA Ladder | ThermoFisher | Cat#12373031 |
Gibson Assembly Master Mix | NEB | Cat#E2611 |
Phusion Flash High-Fidelity PCR Master Mix | ThermoFisher | Cat#F548L |
QIAGEN Plasmid Maxi Kit | QIAGEN | Cat#12162 |
Fixation/Permeabilization Solution Kit | BD | Cat#554714 |
FastDigest BshTI | ThermoFisher | Cat#FD1464 |
FastDigest BstXI | ThermoFisher | Cat#FD1024 |
FastDigest XhoI | ThermoFisher | Cat#FD0694 |
FastDigest KpnI | ThermoFisher | Cat#FD0524 |
CellTiter-Glo® Luminescent Cell Viability Assay | Promega | Cat#G7572 |
SF Cell Line 4D-NucleofectorTM X Kit L | Lonza | Cat# V4XC-2012 |
RNeasy Plus Mini Kit (250) | QIAGEN | Cat# 74136 |
NEBNext Ultra II Directional RNA Library Prep Kit for Illumina | NEB | Cat# E7760S |
NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1) | NEB | Cat# E7335S |
XenoLight D-Luciferin - K+ Salt Bioluminescent Substrate | Perkin Elmer | Cat#122799 |
M-MLV Reverse Transcriptase | Sigma | Cat# 11062603001 |
Deposited Data | ||
Structure of the Nsp1-40S complex | This paper | PDB: 7JQB |
Structure of the Nsp1-40S-CrPV IRES complex | This paper | PDB: 7JQC |
Cryo-EM map of the Nsp1-40S complex | This paper | EMD: 22432 |
Cryo-EM map of the Nsp1-40S-CrPV IRES complex | This paper | EMD: 22433 |
Source data and summary statistics of cellular viability effect by introduction of SARS-CoV-2 viral proteins and mutants | This paper | Table S2 |
Processed Nsp1 mRNA-seq dataset and differential expression analysis | This paper |
Table S3 GEO/SRA accession number: PRJNA667046 |
DAVID pathway analysis of Nsp1 differentially expressed gene sets | This paper | Table S4 |
Experimental Models: Cell Lines | ||
H1299 | ATCC | ATCC® CRL-5803 |
VeroE6 | ATCC | ATCC® CRL-1586 |
Oligonucleotides | ||
All standard cloning primers for Gibson assembly | This paper | Table S1 |
Amplicon primers for cloning | This paper | Table S1 |
ACTB (hs01060665_g1) | Thermo Fisher | Cat#4331182 |
NSP1 Taqman Probe | Invitrogen | In this paper |
Recombinant DNA | ||
pETDUET-1 | EMD Millipore | Cat#71146 |
pMAL system | New England Biolabs | Cat#E8200S |
Plasmid: 6xHis-Nsp1 in pETDUET-1 | This paper | N/A |
Plasmid: 6xHis-MBP-Nsp1 in pMAT9S (lab-made p-MAL derivative) | This paper | N/A |
Plasmid: 6xHis-eIF3j in pETDUET-1 | This paper | N/A |
pcDNA3.1 | Addgene | Cat#52535 |
pVPSB empty | This paper | N/A |
pVPSB-ORFs | This paper | N/A |
Nsp1 mutant1 | This paper | N/A |
Nsp1 mutant3 | This paper | N/A |
Nsp1 mutant4 | This paper | N/A |
Lenti-Fluc-Puro | This paper | N/A |
Software and Algorithms | ||
Phenix (v1.18.2) | Adams et al., 2010 | https://www.phenix-online.org/ |
Coot | Emsley et al., 2010 | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ |
cryoSPARC (v2.15) | Punjani et al., 2017 | https://cryosparc.com/ |
PyMOL (v2.1) | Schrödinger, LLC, 2015 | https://pymol.org/2/ |
Chimera (v1.14) | Pettersen et al., 2004 | https://www.cgl.ucsf.edu/chimera/ |
SerialEM | Mastronarde, 2005 | https://bio3d.colorado.edu/SerialEM/ |
NanoAnalyze (v3.6.0) | TA Instruments | https://www.tainstruments.com/support/software-downloads-support/downloads/ |
Clustal Omega | Sievers et al., 2011 | https://www.ebi.ac.uk/Tools/msa/clustalo/ |
FlowJo software 9.9.6 | FlowJo | https://www.flowjo.com |
Kallisto 0.45.0 | Bray et al., 2016 | https://pachterlab.github.io/kallisto/ |
Sleuth 0.30.0 | Pimentel et al., 2017 | https://pachterlab.github.io/sleuth/about |
DAVID 6.8 | Huang et al., 2009 | https://david.ncifcrf.gov/ |
GSEA 4.0.3 | Subramanian et al., 2005 | https://www.gsea-msigdb.org/gsea/index.jsp |
Other | ||
Ni-NTA Agarose | QIAGEN | Cat#30230 |
HiTrap Q HP 5mL | GE Healthcare | Cat#17115401 |
HiLoad 16/600 Superdex 75 PG | GE Healthcare | Cat#28989333 |
NuPAGE 4-12% Bis-Tris Midi gels | Invitrogen | Cat#WG1403B0X |
Protein Concentrators PES, 100K MWCO | Thermo Scientific | Cat#88503 |
Amicon Ultra-15 Centrifugal Filter Units 10kDa | Millipore Sigma | Cat#UFC901024 |
C-flat Holey Carbon for Cryo-TEM | Electron Microscopy Sciences | Cat#312-50 |